1. Field of the Invention
This invention relates generally to new polypeptides, to methods for preparation of the new polypeptides, and fields of use for the polypeptides.
2. Description of the Prior Art
It is well known that many polypeptides have been isolated from various organs of animals. Until about the past decade, however, very little was known about the thymus, an organ which in man comprises about 0.8% of his body weight at birth, although it has been previously hypothesized that a neuromuscular blocking substance existed in the thymus and that thymic hormone affected the development of the immune system. Despite keen interest in possible functions of the thymus and early speculation and experimentation, little was known of the function of the thymus until recently. It is now realized, however, that the thymus is a compound organ with both epithelial (endocrine) and lymphoid (immunological) components and thus the thymus is involved in the immunity functions of the body. The thymus is known to be a compound organ consisting of an epithelial stroma derived from the third branchial arch and lymphocytes derived from stem cells originating in haemopoietic tissues, Goldstein et al, The Human Thymus, Heinemann, London, 1969. Lymphocytes are differentiated within the thymus and leave as mature thymus-derived cells, called T cells, which circulate to the blood, lymph, spleen, and lymph nodes. The induction of stem cell differentiation within the thymus appears to be mediated by secretions of the epithelial cells of the thymus but difficulties with bioassays had previously hindered the complete isolation and structural characterization of any hormones which may be present.
To provide an understanding of the importance of the differentiating biological characteristics of the polypeptides of this invention, it should be noted that the function of the thymus in relation to immunity may be broadly stated as the production of thymus-derived cells, or lymphocytes, which are called T cells. T cells form a large proportion of the pool of recirculating small lymphocytes. T cells have immunological specificity and are directly involved in cell-mediated immune responses (such as homograft responses), as effector cells. T cells, however, do not secrete humoral antibodies as these antibodies are secreted by cells derived directly from the bone marrow independently of the thymic influence and these latter cells are termed B cells. However, for many antigens, B cells require the presence of appropriately reactive T cells before they can produce antibodies. The mechanism of this process of cell cooperation is not yet completely understood.
From this explanation, it may be said that in operational terms, the thymus is necessary for the development of cellular immunity and many humoral antibody responses and it affects these systems by inducing, within the thymus, the differentiation of haemopoietic stem cells to T cells. This inductive influence is mediated by secretions of the epithelial cells of the thymus, that is, the thymic hormones.
Further, to understand the operation of the thymus and the cell system of lymphocytes, and the circulation of lymphocytes in the body, it should be pointed out that stem cells arise in the bone marrow and reach the thymus by the blood stream. Within the thymus, stem cells become differentiated to immunologically competent T cells, which migrate to the blood stream and together with B cells, circulate between the tissues, lymphatics, and the blood stream.
The cells of the body which secrete antibody also develop from haemopoietic stem cells but their differentiation is not determined by the thymus. Hence, they are termed bone marrow-derived cells or B cells. In birds they are differentiated in an organ analogous to the thymus, which is called the Bursa of Fabricius. In mammals no equivalent organ has been discovered and it is thought that B cells differentiate within the bone marrow. The physiological substances dictating this differentiation remain completely unknown.
It has been known for some time that the thymus is connected with the immunity characteristics of the body and therefore great interest has been indicated in substances which have been isolated from the thymus. In this regard, there have been published in recent years a relatively large body of articles based on scientific work relating to materials which are present in bovine thymus. In fact, the Applicants have published a number of articles which relate to research in this area. Pertinent publications may be found, for example, in The Lancet, July 20, 1968, pps. 119-122; Triangle, Vol. 11, No. 1, pps. 7-14, 1972; Annals of the New York Academy of Sciences, Vol. 183, pps. 230-240, 1971; Clinical and Experimental Immunology, Vol. 4, No. 2, pps. 181-189, 1967; Nature, Vol. 247, pps. 11-14, 1974; Proceedings of the National Academy of Sciences, USA, Vol. 71, pps. 1474-1478; Cell, Vol. 5, pps. 361-365, and pps. 367-370, 1975; and Lancet, Vol. 2, pps. 256-259, 1975.
In the article by Goldstein and Manganaro in Annals of the New York Academy of Sciences, Vol. 183, pps. 230-240, 1971, there are disclosures regarding the presence of a thymic polypeptide which causes a myasthenic neuromuscular block in animals, which is analogous to the human disease of myasthenia gravis. Further, in this article it was discovered that two distinct effects were caused by separate polypeptides in bovine thymus. One of these polypeptides, named "thymotoxin", was believed to cause myositis but it was further indicated that this polypeptide had not been isolated although it appeared to be a polypeptide of approximately 7,000 molecular weight, had a strong net positive charge and was retained on CM-Sephadex at a pH of 8.0.
In the publication "Nature", 247, 11, Jan. 4, 1975, there are described products identified as Thymin I and Thymin II which were found to be new polypeptides isolated from bovine thymus which have particular uses in various therapeutic areas. Because of the use of similar names for other products isolated from the thymus in the prior art, these Thymin I and Thymin II products are now named as Thymopoietin I and Thymopoietin II. These products and processes are described in U.S. Pat. No. 4,077,949, issued Mar. 7, 1978, from Ser. No. 606,843, filed Aug. 22, 1975, which is a continuation-in-part of Application Ser. No. 429,202, filed Dec. 28, 1973, and now abandoned.
In issued U.S. Pat. No. 4,002,602, dated Jan. 11, 1977, which is a continuation-in-part of Application Ser. No. 449,686, filed Mar. 11, 1974, now abandoned, there are disclosed long chain polypeptides described as Ubiquitous Immunopoietic Polypeptides (UBIP), which polypeptide is a 74-amino acid polypeptide characterized by its ability to induce in vitro, in nanogram concentrations, the differentiation of both T-cell and B-cell immunocytes from precursors present in bone marrow or spleen. Thus the polypeptide is useful in therapeutic areas involving thymic or immunity deficiences and the like.
In issued U.S. Pat. No. 4,002,740, issued Jan. 11, 1977 there are disclosed synthesized tridecapeptide compositions which have the capability of inducing the differentiation of T-lymphocytes but not of complement receptor B-lymphocytes. This polypeptide thus exhibited many of the characteristics of the long chain polypeptides isolated and named as thymopoietin in above-mentioned U.S. Pat. No. 4,077,949.
The present invention provides a synthesized five-amino acid polypeptide having a definite active site sequence which which has been found to exhibit many of the characteristics of the long chain polypeptide isolated and name as Thymopoietin in the above publications and U.S. Pat. No. 4,077,949 and the tridecapeptide described in U.S. Pat. No. 4,002,740.